TDP-43 proteinopathy in ALS is triggered by loss of ASRGL1 and associated with HML-2 expression

TAR DNA-binding protein 43 (TDP-43) proteinopathy in brain cells is the hallmark of amyotrophic lateral sclerosis (ALS) but its cause remains elusive. Asparaginase-like-1 protein (ASRGL1) cleaves isoaspartates, which alter protein folding and susceptibility to proteolysis. ASRGL1 gene harbors a copy of the human endogenous retrovirus HML-2, whose overexpression contributes to ALS pathogenesis. Here we show that ASRGL1 expression was diminished in ALS brain samples by RNA sequencing, immunohistochemistry, and western blotting. TDP-43 and ASRGL1 colocalized in neurons but, in the absence of ASRGL1, TDP-43 aggregated in the cytoplasm. TDP-43 was found to be prone to isoaspartate formation and a substrate for ASRGL1. ASRGL1 silencing triggered accumulation of misfolded, fragmented, phosphorylated and mislocalized TDP-43 in cultured neurons and motor cortex of female mice. Overexpression of ASRGL1 restored neuronal viability. Overexpression of HML-2 led to ASRGL1 silencing. Loss of ASRGL1 leading to TDP-43 aggregation may be a critical mechanism in ALS pathophysiology.


Data analysis
The datasets generated during current study have been deposited in the Figshare database under accession "TDP-43 proteinopathy in ALS is triggered by loss of ASRGL1 and associated with HML-2 expression".Public databases have been used for sequence alignment of proteins (PDB, Sprot Human database, Uniprot) and nucleic acids (GRCh38), protein interactions (BioGRID), and gene ontology (Gene Ontology (GO) Consortium).Source data are provided with this paper as Supplementary Data 1.RNA sequencing data was obtained from the ALS Consortium of the New York Genome Center and would available upon request: https:// www.nygenome.org/contact/.
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Randomization
Sex of donors of samples analyzed was always reported (sex assigned at birth).Comparison between ALS samples and controls was always sex matched.See Supplementary tables 1, 2 and 3.
Race and ethnicity have not been reported, since it was not available for most samples.
Demographic characteristics of donors of samples analyzed are described in Supplementary tables 1, 2 and 3.

Ethics oversight
Note that full information on the approval of the study protocol must also be provided in the manuscript Brain samples: kindly provided by the institutions described in Supplementary

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For all in vitro experiments a minimum of 3 independent experimental replicates was performed, which is standard in the field.Sample size for RNA sequencing analysis was determined by the data availability the the NY Genome Center but it meets or exceeds standards in the field (391 samples: 218 males and 173 females).Sample size for immunohistochemistry (IH), western blot (WB) and PCR experiments with brain samples from ALS and controls was chosen according to or exceeding standards in the field: 16 samples for IH, 40 for WB and 39 for PCR.
For the experiments with motor neurons derived from induced pluripotent stem cells lines, we used 12 lines, which also meets and/or exceeds standards in the field.
For the in vivo experiments we used 10 animals (5 per group), which is also standard in the field.Only female mice were used due to limited sample size.
For each in vitro experiment there was a setting up of the protocol.Several trials were performed during this phase whose results were not included in the analysis.Once a protocol was determined to be optimal, the experiment was replicated independently at least 3 times.No data were excluded in the analysis of those replicates.
For the in vitro experiments we always performed between 3 and 7 independent replicates.The results of all replicates were averaged for the final result.
Randomization for in vitro experiments involving cell cultures was not needed.The same cell line was used for the different treatments that were compared in each experiment (transfection with plasmids or peptides or treatment with reagents).For analysis of clinical samples, ALS samples and controls were not randomized; they were paired by sex and age.In microscopy random fields were chosen for counting cells.For in vivo experiments, mice were randomly assigned to receive either AAV vectors encoding ASRGL1 shRNAs or scrambled shRNAS.
Investigators were not blinded for some of the experiments: RNA seq analysis, in vitro experiments involving western blotting, since the same investigator performed each experiment from group allocation to data collection.However, measurements were always confirmed by a second investigator.For experiments involving cellular or tissue staining of human clinical samples or mice, the staining was performed by a non-blinded investigator but the analysis was performed by two independent blinded investigators.Confirm that both raw and final processed data have been deposited in a public database such as GEO.

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Files in database submission
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Table 2 following institutions' approvals and Material Transfer Agreements.Patient characteristics and sources of the samples are described in Supplementary Table 2. Induced pluripotent Stem Cells: Induced pluripotent stem cells (iPSCs) lines were generated from deidentified PBMCs from healthy human donors (Supplementary Table3) and sporadic ALS patients using a Sendai virus method, as previously described.The lines were generated by the National Heart, Lung, and Blood Institute iPSCs core faculty at the National Institutes of Health (NIH).Samples were obtained under protocols 15-N-0125 (ALS patients) and 03-AG-0325 (Normal controls) following approval by the Institutional Review Board of NIH.RNA sequencing data: RNA sequencing data was acquired from the New York Genome Center (NYGC) Consortium Database (https:// www.nygenome.org/contact/)followinginstitution'sapprovaland Material Transfer Agreement.Demographic characteristics of the ALS patients and controls are described in Supplementary Table1.The lines were generated by the National Heart, Lung, and Blood Institute iPSCs core faculty at the National Institutes of Health (NIH).Samples for the generation of Induced pluripotent stem cell lines were obtained under protocols 15-N-0125 (ALS patients) and 03-AG-0325 (Normal controls) following approval by the Institutional Review Board of NIH.RNA sequencing data: RNA sequencing data was acquired from the New York Genome Center (NYGC) Consortium Database (https:// www.nygenome.org/contact/)following institution's approval and Material Transfer Agreement.
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